In 2009, Park et al defined the structural basis of the recognition of LPS by TLR4-MD2 (Park B S, Song D H, Kim H M, Choi B S, Lee H, Lee J O. The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature 2009; 458: 1191-1195; Viriyakosol S, Tobias P S, Kitchens R L, Kirkland T N. MD2 binds to bacterial lipopolysaccharide. J. Biological Chemistry 2001; 276(41): 38,044-38,051).
In brief, MD2 has a hydrophobic pocket that is lined by a charged, hydrophilic entrance. Lipid A (which consists of two phosphorylated glucosamine molecules linked together via an ester bond) binds to the entrance of this pocket. Its lipid chains then enter MD2's hydrophobic pocket. The TLR4-MD2-LPS complex undergoes a conformational change and TLR4 dimerizes. Intracellular signalling follows.
The importance of the saccharide portions of LPS and of electrostatic interactions between LPS and MD2 have recently been reinforced (Meng J et al, MD2 residues TYR 42, ARG 69, ASP 122 and LEU 125 provide species specificity for Lipid IVA. J Biol Chem. DOI: M110.134668; Meng J, Lien E, Golenbock D T. MD2 mediated ionic interactions between Lipid A and TLR4 are essential for receptor activation. J Biol Chem. 2009; 285: 8695-8702).
The most relevant residue is thought to be Tyr102, followed by Lys91, Arg96, Arg106, Asn114 and Ser118. These residues and/or residues in their very close proximity had already been identified as having an important role in the recognition of LPS by MD2 (Park B S, Song D H, Kim H M, Choi B S, Lee H, Lee J O. The structural basis of lipopolysaccharide recognition by the TLR4-MD2 complex. Nature 2009; 458: 1191-1195; Meng J et al, MD2 residues TYR 42, ARG 69, ASP 122 and LEU 125 provide species specificity for Lipid IVA. J Biol Chem. DOI: M110.134668; Meng J, Lien E, Golenbock D T. MD2 mediated ionic interactions between Lipid A & TLR4 are essential for receptor activation. J Biol Chem. 2009; 285: 8695-2).
Bacterial infections and surgical tissue injury trigger the same cell surface receptor-ligand interactions that are based upon TLR4 immuno-modulation. This does not involve a single receptor-ligand interaction. Rather, these pro-inflammatory cytokine responses are mediated by polyvalent receptor-ligand interactions between bacterially derived lipopolysaccharide (LPS) and/or surgically derived hyaluronan fragments and the cell surface TLR4 receptor (Atala A, Irvine D J, Moses M, Shaunak S. Wound healing versus regeneration: Role of the tissue environment in regenerative medicine. MRS Bulletin August 2010; 35: 597-606). The binding affinity of these ligands for this receptor increases exponentially as the number of receptor-ligand interactions increases. Therefore, it is desirable to adapt the concept of polyvalency to novel biomaterial design by making new biomaterials that can modulate tissue injury pathways.
As polyvalency requires multiple and co-operative receptor-ligand interactions, pharmacological intervention will also require new medicines that are based upon molecules that are also capable of multiple and co-operative interactions. This has already been achieved with protein-based medicines, which interact with multiple cell surface receptors with high affinity. For many years, the aim has been to achieve analogous co-operative interactions with synthetic macromolecules. However, it has been found that in biological systems, the use of linear polymers has been much less successful than anticipated. Attempts to use linear polymers have been impeded by:—(1) the structural heterogeneity of the macromolecules used; (2) an inability to control their size and molecular weight characteristics; and (3) the toxic side effects of activating complement and coagulation triggered pathways.
In addition, in the case of linear polymers displaying saccharides, they have a tendency to self-associate and to form micelles because of the amphiphilic characteristics of many polymer-saccharide combinations. In the case of polysaccharides, their structural heterogeneity and the complex nature of the chemistry involved in their preparation has impeded the manufacturing scalability and reproducible synthesis of defined oligosaccharide-like molecules with the appropriate biological properties. In general, many synthetic steps are required, and the polar nature of the chemical intermediates and products make them difficult to purify. These compounds are also difficult to handle because they tend to be hygroscopic syrups, chemically labile, susceptible to rapid microbial degradation, and difficult to process into medicines. These fundamental problems have impaired the scale up manufacture of saccharide based macromolecules for pharmaceutical use.
WO 03/089010 disclosed certain glycodendrimers based on the PAMAM core. Generation three of PAMAM dendrimers conjugated to glucosamine or glucosamine sulfate to form a 3.5 generation dendrimer have been extensively studied.
These molecules have been shown to have very interesting biological activity and low toxicity, and in particular have significant anti-cytokine and anti-chemokine properties.
However, these compounds have not been progressed as pharmaceutical products and have never been dosed to a human because, to date, no way has been identified to commercially and viably manufacture them to a level of “purity” that is suitable for administration to a human. What is more many of the so called “impurities” are very closely related chemical species to the desired species and thus separation of the different entities is very difficult and may not be possible using currently available chromatography and/or filtration based techniques.
Whilst not wishing to be bound by theory it now believed by the present inventors that the chemistry on which PAMAM glycodendrimers is based is inherently incompatible with providing a molecule suitable for pharmaceutical use.
Nevertheless PAMAM glycodendrimers are special molecules because of their biological activity. Whilst many cores exist from which dendrimers can be made, it seems that glycodendrimers made from several alternative cores do not possess the requisite biological properties.
The present inventors believe that, surprisingly, the glycodendrimers described herein are likely to provide suitable biological properties to render them suitable for pharmacological intervention and additionally that the chemistry on which the molecules according to the invention are based is suitable for providing a molecule which can ultimately be used as a pharmaceutical product.